RESEARCH ARTICLE Genetic structure and distribution of Parisotoma notabilis (Collembola) in Europe: Cryptic diversity, split of lineages and colonization patterns Helge von Saltzwedel*, Stefan Scheu, Ina Schaefer Georg August University GoÈttingen, Johann Friedrich Blumenbach Institute of Zoology and Anthropology, GoÈttingen, Germany * [email protected] a1111111111 a1111111111 a1111111111 Abstract a1111111111 a1111111111 Climatic and biome changes of the past million years influenced the population structure and genetic diversity of soil-living arthropods in Europe. However, their effects on the genetic structure of widespread and abundant soil animal species such as the Collembola Parisotoma notabilis remain virtually unknown. This generalist and parthenogenetic species OPEN ACCESS is an early colonizer of disturbed habitats and often occurs in human modified environments. Citation: von Saltzwedel H, Scheu S, Schaefer I To investigate ancient climatic influence and recent distributions on the genetic structure of (2017) Genetic structure and distribution of P. notabilis we analyzed populations on a pan-European scale using three genetic markers Parisotoma notabilis (Collembola) in Europe: differing in substitution rates. The results showed that P. notabilis comprises several genetic Cryptic diversity, split of lineages and colonization patterns. PLoS ONE 12(2): e0170909. doi:10.1371/ lineages with distinct distribution ranges that diverged in the Miocene. Genetic distances of journal.pone.0170909 COI between lineages ranged between 15% and 18% and molecular clock estimates sug- Editor: Xiao-Yue Hong, Nanjing Agricultural gest Late Miocene divergences considering the standard arthropod rate of 2.3% per my. University, CHINA Compared to other soil-living arthropods like oribatid mites, European lineages of P. not- Received: July 21, 2016 abilis are rather young and genetically uniform. The close association with anthropogenic habitats presumably contributed to rapid spread in Europe. Accepted: January 12, 2017 Published: February 7, 2017 Copyright: © 2017 von Saltzwedel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which Introduction permits unrestricted use, distribution, and reproduction in any medium, provided the original The ubiquitous soil arthropod species Parisotoma notabilis (SchaÈffer, 1896) is one of the most author and source are credited. successful species among Collembola being locally abundant in virtually any habitat in the Data Availability Statement: All relevant data are temperate and boreal zone. Populations can reach densities of up to 10,000 and 6,000 individu- within the paper and its Supporting Information als per square meter in forest soils and meadows, respectively, but also typically are present in files. arable fields, pastures, urban soils and caves [1±8], and even in extreme habitats such as open Funding: This study was funded by the Deutsche glacier forelands at high elevation [9]. P. notabilis is the most abundant Collembola species in Forschungsgemeinschaft (SCHE 376/13-1 u. 13- Europe [10] and together with Isotomiella minor (SchaÈffer, 1896) it often represents more than 2). 50% of the total individuals in Collembola communities [2,4]. It is morphologically well Competing Interests: The authors have declared defined [11±14], but exhibits inter-population differences in tolerance to low pH, mechanical that no competing interests exist. disturbances and metal pollution [1,6]. According to stable isotope ratios of 15N/14N it feeds as PLOS ONE | DOI:10.1371/journal.pone.0170909 February 7, 2017 1 / 15 Genetic variation of a generalist springtail in Europe generalist on bacteria, fungi and smaller soil animals including protozoans, nematodes and rotifers [15]. Notably, P. notabilis reproduces via parthenogenesis, no males have been found in natural populations [16] except for a Swedish population where males rarely occur [17]. Wind dispersal [8], the potential to start populations from a single female individual and gen- eralist feeding make this species a fast and successful colonizer of new and disturbed habitats [18±22]. The genetic structure of P. notabilis populations is little known except for one study investi- gating genetic variation within and between European populations [23]. Based on two genetic markers (COI and D2 region of 28S rDNA) they demonstrated that P. notabilis comprises four different lineages in Europe, with low genetic variance within (<3% for COI and zero for D2) but high variance between lineages (21% for COI and <3% for D2). The authors concluded that these four lineages represent `cryptic species' which evolved independently but without morphological differentiation. Deep genetic divergences in soil-living arthropods have been described previously and may be due to strong founder effects and genetic bottlenecks after long-distance dispersal com- bined with limited local dispersal within the soil matrix [24]. Ancient divergences and survival in small patches during the Quaternary Ice-Ages may also generate patterns of deep divergence in P. notabilis, however, these questions have not been addressed yet. In order to investigate the relevance of founder effects and historical dispersal patterns, we extended the geographic sampling of the previous study [23] and included western Russia, the Ukraine, Turkey, the Balkan Peninsula, Norway, Great Britain and Greenland. Thus, the sam- pling included areas of northern Europe that were covered by glaciers during the last Ice Age, i.e. regions that must have been colonized in the Holocene by P. notabilis, resulting in popula- tions of low genetic variation. Colonization likely occurred from southern Europe and south- eastern Russia, similar to the grasshopper Chorthippus parallelus, the hedgehog Erinacues eur- opeaus, the bear Ursus arctos, the alder Alnus glutinosa and oaks Quercus spp. [25±27]. There- fore, we expected northern lineages to be closely related to lineages of western and central Europe, but distantly to those south of the Alps. We used three genetic markers, the mitochondrial COI gene, the D3-D5 region of 28S rDNA and the nuclear gene Histone H3 that provided resolution intermediate to 28S and COI. This is the first study comparing two protein-coding genes and one ribosomal gene with different mutation rates to detect recent and old diversifications, independent evolutionary units (IEUs) and colonization patterns of Collembola in Europe. Materials and methods Ethics statement Sampling sites were outside Nature Reserve Areas and no permission for soil samples was required. The field study did not involve any endangered or protected species. Sampling of animals and DNA extraction Leaf litter and humus layers from about two square meters of deciduous and coniferous forests was collected in 26 locations in Europe, including Greenland, northwest Russia (Karelia), Ukraine, Turkey, the Balkan region (Bulgaria, Serbia, Croatia, Greece), Italy, Spain, and trans- ferred to the University of GoÈttingen (Fig 1, Table 1). Animals were extracted by heat, collected in water [28], transferred into 96% EtOH and stored at -20ÊC until further analyses. For species identification specimens were sorted under a dissecting microscope and determined by light microscopy following [29]. Genomic DNA was extracted from single individuals of P. notabilis (n = 120) using the DNeasy1 PLOS ONE | DOI:10.1371/journal.pone.0170909 February 7, 2017 2 / 15 Genetic variation of a generalist springtail in Europe Fig 1. Sampling locations and distribution of lineages of Parisotoma notabilis. Genetic lineages were named following Porco et al. (2012), lineage L0 (grey triangles) occurs in three sampling locations (DE1, DK, GB1), while lineages L1 (green circles) and L2 are widespread in the southwest and the east of Europe, respectively. Lineages L3 (yellow diamond) and L4 (yellow turned triangle) are geographically isolated in the south of Europe. doi:10.1371/journal.pone.0170909.g001 Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol for animal tissue. Purified DNA was eluted in 30 μl buffer AE and stored at -20ÊC until further preparation. Two nuclear genes, Histone H3 and the D3-D5 region of 28S rDNA, and the barcoding fragment of the mitochondrial COI gene were amplified in 25 μl volumes contain- ing 12.5 μl SuperHot Taq Mastermix (Genaxxon Bioscience GmbH, Ulm, Germany) with 1.5 μl of each primer (10 pM), 4.5 μl H2O, 2 μl MgCl2 (25 mM) and 3 μl template DNA. A 374 bp fragment of the protein coding gene H3 was amplified, using the primers H3F1 5’- ATG GCT CGT ACC AAG CAG ACV GC-3’ and H3R1 5’-ATA TCC TTR GGC ATR ATR GTG AC-3’ [30]. A ~573 bp fragment of the nuclear 28S rDNA was amplified using the primers 28Sa 5’-GAC CCG TCT TGA AGC ACG-3’ and 28Sbout 5’-CCC ACA GCG CCA GTT CTG CTT ACC-3’ [31]. For the 709 bp fragment of the COI gene the primers LCO1490 5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’ and HCO2198 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’ [32] were used. PCR conditions included one initial activation step at 95ÊC for 15 min, followed by 35 amplification cycles of denaturation at 94ÊC for 15 s, annealing at 45ÊC (COI) or 49ÊC (28S) or 59ÊC (H3) for 15 s, elongation at 72ÊC for 15 s and a final elon- gation step at 72ÊC for 6 min. Positive PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, eluted in 30 μl HPLC water and sent for direct sequencing to the GoÈttingen